The long-term goal of this proposal is to define the immune function of dendritic cells (DC) in the lung. The observations that DC express tight junction (TJ) proteins and metalloproteinases, and that the claudins, a family of TJ proteins, can regulate metalloproteinase activity prompts the following three questions central to the understanding of lung DC biology. 1. How do DC maintain the TJ harrier as they traverse the paracellular pathway to sample pathogens in the airways? To answer this question the structure/function of TJs formed between DC and epithelial cells is assessed in vitro during chemokine induced migration of DC across murine airway epithelial cell line monolayers and in vivo following antigen inhalation. 2. Do the metalloproteinases MMP-2 and/or MMP-9 facilitate DC migration through TJs? This is addressed by examining DC in MMP2-/- or MMP-9-/- mutant mice, in vivo to determine the role of MMP-2 and/or MMP-9 in facilitating the migration of DC through TJs. Using short interfering RNAs, studies in vitro will examine the role of claudin-1 in regulating MT1- MMP mediated activation of MMP-2. 3. Do DCs play a critical role in the granulomatous immune response by releasing MMP-2 and/or MMP-9, which in turn activate cytokines and promote DC migration to local lymph nodes? To answer this question antigen-coated beads are administered to MMP-2-/- or MMP-9-/- mutant mice to induce granuloma formation. The size of the granuloma and the number of contained DC are monitored over time. Laser capture microdissection combined with real time quantitative PCR is used to examine the cytokine milieu within the granulomas. These studies will contribute important new insights into the role of pulmonary DC in the immune defense of the lung. The information obtained will assist in devising new strategies to defend against inhaled pathogens.